Little is known about regulation of mineralocorticoid receptor (MR) function by protein kinases. However, kinases regulate the ability of other steroid receptors to bind hormone, translocate to the nucleus, bind to DNA and alter transcription. New experimental tools now allow the effects of kinases on MR function to be defined. We have generated an anti-MR antibody which recognizes all forms of the MR. Since no mammalian cell line expresses MR and retains physiologic responsiveness to aldosterone (ALDO), methods to isolate colonic crypt and surface cells have been developed. In both cells ALDO is required for MR nuclear translocation. Atrial natriuretic peptide (ANP) and cGMP decrease MR steroid binding and inhibit MR nuclear translocation in Na-absorbing surface cells but not in Cl-secreting crypt cells. Conversely, cGMP increases steroid-bound MR in crypt cells. Epidermal growth factor (EGF) induces nuclear translocation of unliganded MR in crypt but not in surface cells. This suggests that regulation of MR function by kinases is cell- specific. The aims of this proposal are (1) to determine if unliganded and liganded MR are associated with the 90 kDa, 70 kDa and 56 kDa heat shock proteins (hsp) since interaction of hsps with other steroid receptors is an important determinant of receptor function, (2) to characterize the interaction between ANP and the MR by determining if ANP increases cGMP and stimulates cGMP-dependent protein kinase, if ANP decreases MR protein or mRNA, if ANP disrupts the interaction of MR with the nuclear transport machinery, if ANP alters the association of MR with hsps, if stimulation of cGMP by other pathways mimics the effects of ANP on the MR, and if ANP inhibition of MR function inhibits ALDO induction of conductive Na transport, (3) to assess if EGF receptor and other kinases known to regulate other steroid receptors affect MR steroid binding, DNA binding and intracellular distribution, and (4) to compare regulation of MR and glucocorticoid receptor function within the same cell. Surface and crypt cells will be isolated from rat distal colon. Heat shock proteins will be detected with specific antibodies by immunoprecipitation and Western blotting. MR nuclear translocation will be determined by indirect immunofluorescence using an anti-MR antibody and confocal laser scanning microscopy. Whole cell [3H]ALDO binding assay with calculation of Bmax and Kd by Scatchard analysis will assess the effect of kinases on MR steroid binding. MR immunoreactive protein will be measured by quantitative immunoblots, MR mRNA by ribonuclease protection assay and MR activation by mobility shift assay. These studies will evaluate the role of kinases in MR function. This knowledge is critical to understanding how ALDO responsiveness is controlled at the cellular level and potential disorders of this regulation in pathophysiologic states. For example, abnormalities in MR function in vascular smooth muscle or kidney might contribute to the pathogenesis of hypertension or inhibition of MR function by elevated ANP levels common in diabetes may cause the ALDO resistance observed in this condition.